The thymus produces an abundance of MHC restricted, self-tolerant CD4 helper and CD8 cytolytic T cells to participate in adaptive immune responses. Such ?conventional T cells? develop in response to low-affinity TCR interactions with MHC molecules that display a diversity of self-peptides in the cortical region of the thymus. In contrast, high affinity interactions result in clonal deletion, but can also support the survival and differentiation of mature regulatory effectors in some contexts. These effectors include foxP3+ natural regulatory T cells (Treg), intraepithelial lymphocytes (IEL), and CD1d restricted natural killer T cells (NKT). This proposal will explore the role of high-affinity TCR signaling in clonal deletion and NKT effector differentiation. In the previous period we developed a novel approach to capturing T cell clones that are normally destined for deletion. Here we apply this approach to study the repertoire, reactivity, and structure of clones deleted at the early (DP) stage versus those deleted at the late (SP) stage. Specifically we hypothesize that early deleted clones have a distinct interaction with MHC that is less dependent on peptide, whereas late deleted clones have classic peptide-MHC reactivity. Experiments are proposed that will characterize the distinct autoimmune pathologies created by the escape of early versus late deleted clones. Lipid-specific natural killer T cells (NKT) are derived from thymic progenitors that survive strong signaling in the thymus and develop into 3 major effector subsets that produce distinct cytokines, as we showed in the previous period. One of these subsets (NKT2) produces IL-4 in the steady state and this influences multiple aspects of immune cell development to promote type II immune responses. Our research will identify key molecules controlling the differentiation of NKT cells; further characterize the effects of NKT2 produced IL-4 on lymphoid, myeloid, and stromal cells; and define how NKT cells traffic to unique tissue environments to impact immune responses.